Clot Club #5

Factor XIII

Factor XIII Quick Facts

Molecular mass: 320 000 D

Synthesis: Liver, Megakaryocytes

Plasma concentration: 2mg/l

Half-life: 8 days

Normal range: 50 – 150% or 0.5 – 1.5U/ml

Factor XIII, also known as fibrin stabilizing factor, is a heterodimer (FXIII-A2B2) composed of two catalytic A-subunits and two carrier B-subunits. With thrombin cleavage of the A-subunit followed by dissociation of the B-subunit in the presence of calcium, the active site is exposed in the A-subunit. Activated FXIII (FXIIIa) is a transglutaminase that links or ligates the fibrous scaffold consisting of staggered fibrin units. Transglutaminase enzymes may be the earliest clotting enzymes in evolution, but, in vertebrate blood, conversion of FXIII to FXIIIa is tightly regulated. FXIIIa also crosslinks inhibitors of fibrinolysis to fibrin, such as alpha 2-antiplasmin. FXIIIa stabilizes the fibrin clot, and is essential for a properly functioning fibrin matrix.

Biochemistry of Factor XIII (FXIII)

Factor XIII (also known as Fibrin stabilizing factor) is the proenzyme (zymogen) of a glutaminase, which is activated by thrombin. The active factor XIIIa covalently crosslinks the α and β chains thus stabilizing the fibrin network, which develops after the thrombin-mediated cleavage of fibrinogen. The thrombus is thus protected against a premature lysis. At the same time, factor XIIIa mediates the binding of the plasmin inhibitor (α2-antiplasmin) and fibronectin to fibrin. Factor XIII is found in plasma, in thrombocytes and certain tissues (e.g. placenta). In plasma factor XIII is present as a tetramer (A2B2), in which the active site is localized within the A chain. Clinical significance of Factor XIII Hereditary factor XIII deficiency is extremely rare. An acquired deficiency is observed as a consequence of hepatocyte dysfunction and during asparaginase therapy. In addition, consumption and loss coagulopathy, sepsis, leukemia and acute venous thromboembolism can induce a factor XIII deficiency.

Factor XIII Deficiency

While studies elucidating the role of FXIII in clotting began in the 1940s, it wasn’t until 1966 that the first family with congenital FXIII deficiency was identified. As FXIII is essential for stabilizing the fibrin matrix, the loss of its activity would result in hemorrhage. FXIII deficiency is a very rare disease and most cases are due to mutations causing loss of the catalytic A subunits, but there are a few cases that result in deficiency due to a lack of the B carrier subunit. Congenital deficiency is an autosomal recessive disorder with an estimated incidence of around 1 in 2 million. Although clotting might be normal and thus many of the commonly used laboratory clotting tests remain normal (ie, prothrombin time [PT] and activated partial thromboplastin time [aPTT]), a hemorrhagic condition occurs because of the lack of cross-linking during coagulation. The severity of bleeding can range from very mild to life-threatening, and other conditions can result as well, such as habitual abortion in women. Cases of acquired FXIII deficiency also exist, and although also rare, inhibitors block FXIII activity by various mechanisms. Early diagnosis is critical as the onset can be sudden and severe. These patients require replacement of the FXIII. While reports vary on the FXIII levels that will cause symptoms, it is clear that very low FXIII levels are especially harmful. There is ongoing work on using recombinant FXIII, and at least 1 product is approved for use in the United States. It should be noted that FXIII has a wide range of protein targets, suggesting additional important roles in health and disease.

Factor XIII Assays

Assays for FXIIIa coincide with the ongoing discovery that FXIII plays in the coagulation system. Urea-based assays played a role in understanding the need for this component in clot stabilization, as a formed clot was degraded in certain concentrations of urea without it. The urea-solubility assay subsequently was used to find the first family with FXIII deficiency. As the properties of the protein became clearer, assays based on its mechanism of assay were employed. It was recognized that small amines, such as glycine ethylester, played a role in fibrin stabilization, and that FXIIIa was specific towards amine substrates. Amine incorporation thus became a powerful tool in studying FXIIIa quantitation. When FXIIIa attaches glycine ethylester to a specific peptide substrate, ammonia is released. Various methods have been available for determining ammonia content and this has been utilized for many years in different assays. In these colorimetric assays, released ammonia is monitored in an NAD(P)H dependent glutamate dehydrogenase (GlDH) reaction. The decrease in absorbance measured at 340 nm over time is proportional to FXIIIa activity. NADH and NADPH absorb at 340 nm whereas NAD and NADP do not. The use of NADH or NADPH as a cofactor in the reaction varies depending on the assay. Spontaneous breakdown of NAD(P)H can occur but this can be compensated by using a reagent blank (using iodoacetamide). In addition, any compounds present in the sample that react with NAD(P)H under the conditions of this assay can give discrepant results for determination of the ammonia concentration. This side reaction can be as a result of the presence of other enzymes that utilize NADH; however, the use of NADPH as a cofactor can eliminate some of these interferences, as in the case of lactate dehydrogenase (LDH) which cannot utilize NADPH. With use of a blank and in an NADPH-dependent reaction, the sensitivity can be as low as 0.6%.

And now for (some) of the rest of the story of FXIII:

FXIII is also known as Laki–Lorand factor, after Kalman Laki and Laszlo Lorand, the scientists who first proposed its existence in 1948. Factor XIII levels are not measured routinely, but may be considered in subjects with an unexplained bleeding tendency. The Prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen assay are not affected by the absence of FXIII.

Therefore, when you have a subject who is suffering from a bleeding episode and the normal tools for detecting the absence of blood coagulation factors FI-FXII you have to look at the history and go one step further and look for the presence of a FXIII deficiency.

Usually only large reference laboratories or special coagulation laboratories are equipped to perform FXIII quantitative deficiency and inhibitor testing.

The smaller laboratory can perform a qualitative screening method. This clot solubility test uses 5M urea and/or monochloroactetic acid (MCA). This is an option, however, these assays are not now currently recommended by the International Society of Hemostasis and Thrombosis (ISTH) Scientific and Standardization Committee.

Verifying a FXIII deficiency is difficult. As recently as 2016 an article by A Dorghalaleh et al: in Lab Med performed a comparison of 2 methods of clot solubility testing in detection of FXIII deficiency In Iran. This is a country that has few options and limited resources for laboratory testing. However, they have a FXIII deficiency rate that is 12 times greater than the rest of the world. This makes the ability to detect FXIII issues a serious problem. Therefore, the ability to use a screening assay is extremely important.

The research study evaluated 83 patients with normal routine coagulation tests. Twenty nine patients with 5M Urea and 21 with MCA were abnormal; 18 subjects were abnormal with both methods; 2 were abnormal with the MCA but normal with the 5M Urea;10 patients had positive results with 5M Urea but normal results with MCA.

The study group found that the best method for screening for a FXIII deficiency was the 5M urea method using thrombin as the clotting substance that was the most sensitive. They found that 90.5% of the subjects with a FXIII deficiency were detected with the 5M Urea method. However, they contended that the possibility of a 10% misdiagnosis could yield a high incidence of morbidity and mortality. The simultaneous use of both solubility methods could prevent a misdiagnosis. Other studies have said that the solubility tests can only detect extremely low levels of FXIII. Other quantitative more sensitive methods should be performed when an abnormal result is found with the solubility assays.1

Despite being discovered in 1948 it wasn’t until 1960 that the clinical issue of a clinical FXIII deficiency was first described by Duckert F, June F, Shimerling DH about a previously undescribed congenital bleeding abnormality due to a lack of fibrin stabilizing factor.2 The case involved a young boy with who had a severe bleeding diathesis with normal screening coagulation testing but a soluble clot breakdown with 5M urea that was corrected by mixing with normal plasma.

Despite this rare autosomal recessive defect first being described in 1960 it was not until 2011 that the ISTH Scientific and Standardization Committee (SSC) on FXIII and fibrinogen was published as an official SSC communication that the FXIII disorders were classified.3 They determined that inherited FXIII deficiencies can occur from FXIII-A or FXIII-B deficiency. When a severely decreased FXIII-A deficiency occurs the level of FXIII-B levels can be modestly reduced to normal range levels. In the presence of rare severely decreased FXIII-B levels FXIII-A is considerably reduced, due to accelerated clearance but does not reach the low levels of a congenital FXIII-A deficiency.3 Factor XIII-A has two subtypes I and II. The two subtypes denote quantitative and qualitative defects of FXIII-A.

There are several methods currently in use for the diagnosis of a FXIII deficiency but not all laboratories have the capability to perform them. These assays include the already discussed clot-solubility methods, quantitative FXIII activity assays and FXIII antigen tests for the FXIII complex A2B2 and for subunits FXIII-A and FXIII-B. The clot solubility assay however can only detect severely decreased levels of FXIII. The FXIII quantitative assays being performed for FXIII activity and antigen is increasing as testing is becoming standardized due to organizations such as the ISTH/SSC committees recommending their use. The use of standardized specimens by organizations such as the North American Specialized Coagulation Laboratory Association (NASCOLA) for North American Laboratories and close collaboration with the European-based organization External Quality Control of Diagnostic Assays and Tests (ECAT), an international proficiency-testing program that focuses on thrombosis and hemostasis, has encouraged coagulation laboratories to go to the more sophisticated testing to aid in the diagnosis of FXIII-deficiency. This has helped laboratories compare the use of various methods and their interpretation of testing using these standardized specimens.

The ISTH/SSC in 2011 produced a recommended algorithm for the diagnosis of FXIII deficiencies and inhibitors of FXIII. (1) If all normal screening tests are negative but bleeding persists perform quantitative functional FXIIIa activity assay (recommended screening assay for all forms of FXIII deficiency. (2) If abnormal perform the plasma FXIII ELISA studies or platelet lysate activity and ELISA. This will discriminate quantitative vs qualitative defects and determine which subunits are deficient. Normal platelet lysate values suggest either B-subunit deficiency or the presence of anti-FXIII antibodies. (3) Run activity mixing studies and bleeding assays, The tests will evaluate for the presence of neutralizing antibodies (mixing studies) and non-neutralizing antibodies (binding assays. (4) SDS-PAGE Fibrin Crosslinking Analysis will verify the presence of FXIII-dependent fibrin crosslinking. (5) Genetic testing will detect sequencing of F13A1and F13B genes to determine the presence of absence of causative mutations4.

Not all laboratories can perform these assays. We have already discussed the clot solubility assays and discussed their disadvantages and advantages. Then there are the quantitative activity assays. These are based on two methods: the transglutaminase activity and the isopeptidase activity.

Ammonia-release assays: The most commonly used in the clinical laboratory for quantification of FXIII. It is an indirect measurement of transglutaminase (TG) activity. Ammonia released by the TG reaction is measured in a side reaction using a spectrophotometer to quantify ammonia-dependent reduction of NAD(P)H. These tests are easy to automate. Their disadvantage is that some have relatively low sensitivities to levels below <0.05 IU mL. For best results a plasma blank must be used to correct the presence of iodoacetamide (potent FXIII antagonist. If the method does not use as plasma blank overestimation of the FXIIIa activity by 0.02-0.15 IU mL. However, the FXIIIa ammonia-release methods are not to date approved in all countries.4

Amine-incorporation assays: These methods quantify the TG of isoamide bonds catalyzed by FXIIIa. These methods are extremely sensitive but are labor and time intensive in comparison to the ammonia-release assays and are not easy to automate.

Isopeptidase Assay: This method of FXIIIa-dependent hydrolysis of isopeptide bods was discovered in 1997 this procedure employs a fluophore and a fluorescent quencher linked by gamma/epsilon bonds and the fluorescence is read by a fluorometer. However this method has in some studies not compared to the gold-standard ammonia-release methods.

Factor XIII quantitative antigen methods are considered second line or follow-up tests by the ISTH-SSC.4,5 The antigen assays may be used for the detection and classification of FXIII deficiencies and for monitoring response to therapy to include immunoassays measuring FXIII-A2, FXIII-B2 and the FXIII-A2B2 complex. There are various antigenic methods, but are not being used throughout the world due to lack of commercial availability and regulatory approval depending on different parts of the world.5

FXIII Genotyping: This testing is only available for FXIII mutation identification in a few laboratories in the United States for both F13A1 and F13B. Some only offer the FXIII-A Va; 34-> Leu variant. In areas of consanguinity the FXIII-deficiency and mutations are the most prevalent. This was particularly prevalent in in the Southeast Iranian population.5